Download Chapter 8 Bacterial Genetics

Chapter 08
Lecture Outline
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1
A Glimpse of History
 Barbara McClintock (1902–1992)
• Observed that kernel colors in corn not inherited in a
predictable manner
• Concluded segments of DNA moved in and out of genes
involved in color
• Destroyed function of genes
• Published results in 1950
• Met with skepticism
• DNA believed stable
• Idea established by 1970s
• Awarded Nobel Prize in 1983
• Termed transposons
• “Jumping genes”
Antibiotic Resistance
 Staphylococcus aureus
• Gram-positive coccus; commonly called Staph
• Frequent cause of skin and wound infections
• Since 1970s, treated with penicillin-like antibiotics
• For example, methicillin
• In 2004, over 60% of S. aureus strains from
hospitalized patients were resistant to methicillin
• ~2.3 million healthy people in U.S. harbor methicillinresistant S. aureus (MRSA)
• Healthcare-associated MRSA (HA-MRSA) resistant to
other antibiotics, including vancomycin
• Vancomycin considered drug of last resort
8.1. Genetic Change in Bacteria
 Organisms adapt to changing environments
• Natural selection favors those with greater fitness
• Bacteria adjust to new circumstances
• Regulation of gene expression (Chapter 7)
• Genetic change (Chapter 8)
• Bacteria excellent system for genetic studies
• Rapid growth, large numbers
• More known about E. coli genetics than any other
• Change in organism’s DNA alters genotype
• Sequence of nucleotides in DNA
• Bacteria are haploid, so only one copy, no backup
• May change observable characteristics, or phenotype
• Also influenced by environmental conditions
8.1. Genetic Change in Bacteria
 Mutation and horizontal gene transfer
8.1. Genetic Change in Bacteria
 Mutations can change organism’s phenotype
• Deletion of gene for tryptophan biosynthesis yields
mutant that only grows if tryptophan supplied
• Growth factor required; mutant termed auxotroph
– Auxo = “increase”; troph = “nourishment”
• Prototroph does not require growth factors
– Proto = “first”
• Geneticists compare mutants to wild type
• Typical phenotype of strains isolated from nature
• For example, wild-type E. coli strain is prototroph
• Strains designated by three-letter abbreviations
– for example, Trp– cannot make tryptophan
– Streptomycin resistance designated StrR
8.2. Spontaneous Mutations
 Spontaneous mutations caused by normal processes
 Occur randomly at infrequent characteristic rates
• Mutation rate: probability of mutation each cell division
• Typically between 10–4 and 10–12 for a given gene
• Mutations passed to progeny
• Occasionally change back to original state: reversion
• Large populations contain mutants (for example, cells in
colony)
• Environment selects cells that grow under its conditions
•
•
•
•
For example, antibiotics select for resistant bacteria if present
Environment does not cause mutations
Single mutation rare; two even rarer
Physicians may use two antibiotics to reduce resistance
– Chance is product of mutation rate for each
8.2. Spontaneous Mutations
 Base substitution
most common
• Incorrect nucleotide
incorporated during
DNA synthesis
• Point mutation is
change
of a single base pair
8.2. Spontaneous Mutations
 Base substitution leads to three possible
outcomes
• Silent mutation: wild-type amino acid
• Missense mutation: different amino acid
• Resulting protein may only partially function
– Termed leaky
• Nonsense mutation:
Specifies stop codon
– Yields shorter protein
8.2. Spontaneous Mutations
 Base substitutions
• A mutation that inactivates gene is termed a null or
knockout mutation
• “Silent mutation” sometimes used to indicate mutation
that does not alter protein function
• Base substitutions more common in aerobic
environments
• Reactive oxygen species (ROS) produced from O2
• Can oxidize nucleobase guanine
– DNA polymerase often mispairs with adenine
8.2. Spontaneous Mutations
 Deletion or addition of nucleotides
• Impact depends on number of nucleotides
• Three pairs changes one codon
• One amino acid more or less
• Impact depends on location
within protein
• One or two pairs yields
frameshift mutation
• Different set of codons translated
• Often results in premature
stop codon
• Shortened, nonfunctional protein
• Knockout mutation
8.2. Spontaneous Mutations
 Transposons (jumping genes)
• Can move from one location to another
• Process is transposition
• Gene insertionally inactivated
• Function destroyed
• Most transposons have transcriptional terminators
• Blocks expression of downstream genes in operon
8.2. Spontaneous Mutations
 Transposons (jumping genes) (continued…)
• Classic studies carried out by Barbara McClintock
• Observed color variation in corn kernels resulting from
transposons moving into and out of genes controlling
pigment synthesis
8.3. Induced Mutations
 Induced mutations result from outside influence
• Agent that induces change is mutagen
• Geneticists may use mutagens to increase mutation rate
• Two general types: chemical, radiation
8.3. Induced Mutations
 Chemical mutagens may cause base substitutions
or frameshift mutations
 Some chemicals modify nucleobases
• Change base-pairing properties
• Increase chance of incorrect nucleotide incorporation
• Nitrous acid (HNO2) converts cytosine to uracil
– Base-pairs with adenine instead of guanine
8.3. Induced Mutations
 Some chemicals modify nucleobases (cont…)
• Alkylating agents add alkyl groups onto nucleobases
• Nitrosoguanidine adds methyl group to guanine
– Base-pairs with thymine
8.3. Induced Mutations
 Base analogs resemble nucleobases
• Have different hydrogen-bonding properties
• Can be mistakenly incorporated by DNA
polymerase
• 5-bromouracil resembles
thymine, often base-pairs
with guanine
2-amino purine resembles
adenine, often pairs with
cytosine
8.3. Induced Mutations
 Intercalating agents cause frameshift mutations
• Flat molecules that intercalate (insert) between adjacent
base pairs in DNA strand
• Pushes nucleotides apart, produces space
• Causes errors during replication
• If in template strand, a base pair added to synthesized
strand
• If in strand being synthesized, a base pair deleted
• Often results in premature stop codon
• Ethidium bromide is common intercalating agent
• Likely carcinogen
• Chloroquine, used to treat malaria, is another example
8.3. Induced Mutations
 Transposition
• Transposons can be used to generate mutations
• Transposon inserts into cell’s genome
• Generally inactivates gene into which it inserts
8.3. Induced Mutations
 Radiation: two types
• Ultraviolet irradiation forms thymine dimers
• Covalent bonds between adjacent thymines
– Cannot fit into double helix; distorts molecule
– Replication and transcription stall at distortion
– Cell will die if damage not repaired
– Mutations result from cell’s SOS repair mechanism
• X rays cause single- and
double-strand breaks in DNA
– Double-strand breaks
often produce lethal
deletions
• X rays can alter nucleobases
8.4. Repair of Damaged DNA
 Enormous amount of spontaneous and mutageninduced damage to DNA
• If not repaired, can lead to cell death; cancer in animals
• For example, in humans, two genes associated with breast
cancer code for DNA repair enzymes; mutations in either
result in 80% probability of breast cancer
• Mutations are rare; alterations in DNA generally repaired
before being passed to progeny
• Several different DNA repair mechanisms
8.4. Repair of Damaged DNA
8.4. Repair of Errors in Nucleotide Incorporation
 During replication, DNA polymerase sometimes
incorporates wrong nucleotide
• Mispairing slightly distorts DNA helix
• Recognized by enzymes
• Mutation prevented by repairing before DNA replication
• Two mechanisms: proofreading, mismatch repair
• Proofreading by DNA polymerase
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•
•
•
Verifies accuracy
Can back up, excise nucleotide
Incorporate correct nucleotide
Very efficient but not flawless
8.4. Repair of Errors in Nucleotide Incorporation
• Mismatch Repair
• Fixes errors missed by DNA
polymerase
• Enzyme cuts sugarphosphate backbone
• Another enzyme degrades
short region of DNA strand
• Methylation of DNA indicates
template strand
– Methylation takes time,
so newly synthesized
strand is unmethylated
• DNA polymerase, DNA ligase
make repairs
8.4. Repair of Modified Nucleobases in DNA
 Modified nucleobases lead to
base substitutions
• Glycosylase removes
oxidized nucleobase
• Another enzyme cuts
DNA at this site
• DNA polymerase
removes short section;
synthesizes replacement
• DNA ligase seals gap
8.4. Repair of Thymine Dimers
 Several methods to repair damage from UV light
• Photoreactivation: light repair
• Enzyme uses energy from light
• Breaks covalent bonds of
thymine dimer
• Only found in bacteria
• Excision repair: dark repair
• Enzyme removes damage
• DNA polymerase, DNA ligase
repair
Repair of Thymine Dimers
 Several methods to repair damage from UV light
(continued…)
• SOS repair: last-ditch repair mechanism
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•
•
•
Induced following extensive DNA damage
Photoreactivation, excision repair unable to correct
DNA and RNA polymerases stall at unrepaired sites
Several dozen genes in SOS system activated
– Includes a DNA polymerase that synthesizes even in
extensively damaged regions
– Has no proofreading ability, so errors made
– Result is SOS mutagenesis
8.5. Mutant Selection
 Mutants rare, difficult to isolate
• Two main approaches
• Direct selection: cells inoculated onto medium that
supports growth of mutant but not parent
• for example, antibiotic-resistant mutants exposed to
antibiotic
• Indirect selection: isolates
auxotroph from prototrophic
parent strain
• More difficult since parents will
grow on any media on which
auxotroph can grow
• Replica plating
8.5. Mutant Selection
8.5. Mutant Selection
 Penicillin enrichment of mutants sometimes
used
• Increases proportion of auxotrophs in broth culture
• Penicillin kills growing cells
• Prototrophs
• Auxotrophs survive
• Penicillinase then added
• Cells plated on rich medium
8.5. Mutant Selection
 Screening for Possible Carcinogens
• Carcinogens cause many cancers; most are mutagens
• Animal tests expensive, time-consuming
• Mutagens increase low frequency of spontaneous
reversions
• Ames test measures effect of chemical on reversion
rate of histidine-requiring Salmonella auxotroph
• Uses direct selection
• If mutagenic, reversion rate increases relative to control
• Rat liver extract added since non-carcinogenic chemicals
often converted to carcinogens by animal enzymes
• Additional tests on mutagenic chemicals to determine if
carcinogenic
8.5. Mutant Selection
Horizontal Gene Transfer as a Mechanism
of Genetic Change
 Microorganisms commonly acquire
genes from other cells: horizontal gene
transfer
• Can demonstrate recombinants
with auxotrophs
• Combine two strains
– for example, His–, Trp– with Leu–, Thr–
• Spontaneous mutants unlikely
• Colonies that can grow on
glucose-salts medium most likely
acquired genes from other strain
Horizontal Gene Transfer as a Mechanism
of Genetic Change
 Genes naturally transferred by three mechanisms
• Transformation: naked DNA uptake by bacteria
• Transduction: bacterial DNA transfer by viruses
• Conjugation: DNA transfer during cell-to-cell contact
Horizontal Gene Transfer as a Mechanism
of Genetic Change
 DNA replicated only if replicon
• Has origin of
replication
• Plasmids,
chromosomes
• DNA fragments
added to
chromosome via
homologous
recombination
• Only if sequence
similar to region
of recipient’s
genome
8.6. DNA-Mediated Transformation
• Naked DNA is not within
cell or virus
• Cells release when lysed
• Addition of DNase
prevents transformation
• Demonstration of
transformation in
pneumococci
• Only encapsulated cells
pathogenic
8.6. DNA-Mediated Transformation
 Transformation
• Recipient cell must be competent
• Most take up regardless of origin
• Some accept only from closely
related bacteria (DNA sequence)
• Process tightly regulated
• Bacillus subtilis has twocomponent regulatory system
• Recognizes low nitrogen or
carbon
• High concentration of bacteria
(quorum sensing)
• Only a fraction of population
becomes competent
8.7. Transduction
 Transduction: transfer of genes by
bacteriophages
• Specialized transduction: specific genes (Chapter 13)
• Generalized transduction: any genes of donor cell
• Rare error
during phage
assembly
• Transfer of
DNA to new
bacterial host
8.8. Conjugation
 Conjugation: DNA transfer between bacterial cells
• Requires contact between donor, recipient cells
• Conjugative plasmids direct their own transfer
• Replicons
• F plasmid (fertility)
of E. coli most
studied
• Other plasmids encode
resistance to some antibiotics
• Spread resistance easily
8.8. Conjugation
 Conjugation (continued…)
• F plasmid of E. coli
• F+ cells have, F– do not
• Encodes proteins including
F pilus
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•
•
•
•
Sex pilus
Brings cells into contact
Enzyme cuts plasmid
Single strand transferred
Complementary strands
synthesized
• Both cells are now F+
8.8. Conjugation
 Chromosomal DNA transfer
less common
• Involves Hfr cells (high
frequency of recombination)
• F plasmid is integrated into
chromosome via homologous
recombination
• Process is reversible
• F′ plasmid results when small
piece of chromosome is
removed with F plasmid DNA
• F′ is replicon
8.8. Conjugation
 Chromosomal DNA transfer less
common (continued…)
• Hfr cell produces F pilus
• Transfer begins with genes on one
side of origin of transfer of plasmid
(in chromosome)
• Part of chromosome transferred to
recipient cell
• Chromosome usually breaks before
complete transfer (full transfer would
take ~100 minutes)
• Recipient cell remains F– since
incomplete F plasmid transferred
8.9. The Mobile Gene Pool
 Genomics reveals surprising variation in gene
pool of even a single species
• Perhaps 75% of E. coli genes found in all strains
• Termed core genome of species
• Remaining make up mobile gene pool
• Plasmids, transposons, genomic islands, phage DNA
8.9. The Mobile Gene Pool
 Plasmids found in many bacteria and archaea
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•
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•
Some eukarya
Usually dsDNA with origin of replication
Generally nonessential; cells survive loss
Few to many genes
Low-copy-number
• One or a few per cell
• High-copy-number
• Many up to 500
• Most have narrow host range
• Single species
• Some broad host range
• Includes Gram– and Gram+
8.9. The Mobile Gene Pool
 Resistance plasmids (R plasmids)
• Resistance to antimicrobial medications, heavy
metals (mercury, arsenic)
• Compounds found in hospital environments
• Often two parts
• R genes
• RTF (resistance
transfer factor)
– Codes for conjugation
• Often broad host range
• Normal microbiota can
transfer to pathogens
Copyright © 2016 McGraw-Hill Education. Permission required for reproduction or display.
Use fig. 8.26 in 8th edition
8.9. The Mobile Gene Pool
 Transposons provide mechanism for moving DNA
• Can move into other replicons in same cell
• Simplest is insertion sequence (IS)
• Encodes only transposase enzyme, inverted repeats
• Composite transposons include one or more genes
• Integrate via non-homologous recombination
8.9. The Mobile Gene Pool
 Transposons yielded vancomycin resistant
Staphylococcus aureus strain
• Patient infected with S. aureus
• Susceptible to vancomycin
• Also had vancomycin resistant
strain of Enterococcus faecalis
• Transferred transposoncontaining plasmid to S. aureus
• Transposon jumped to
plasmid in S. aureus
8.9. The Mobile Gene Pool
 Bacteria can conjugate with plants
• Natural genetic engineering
• Agrobacterium tumefaciens causes crown gall
• Different properties, produces opine, plant hormones
• Piece of tumor-inducing (Ti) plasmid called T-DNA
(transferred DNA) is transferred to plant
• Incorporated into plant chromosome via nonhomologous recombination
8.9. The Mobile Gene Pool
 Genomic islands: large DNA segments in genome
• Originated in other species
• Nucleobase composition very different from genome
• G-C base pair ratio characteristic for each species
• May provide different characteristics
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Utilization of energy sources
Acid tolerance
Development of symbiosis
Ability to cause disease
– Pathogenicity islands